Distinct expression patterns of different subunit isoforms of the V-ATPase in the rat epididymis.

In the epididymis and vas deferens, the vacuolar H(+)ATPase (V-ATPase), located in the apical pole of narrow and clear cells, is required to establish an acidic luminal pH. Low pH is important for the maturation of sperm and their storage in a quiescent state. The V-ATPase also participates in the acidification of intracellular organelles. The V-ATPase contains many subunits, and several of these subunits have multiple isoforms. So far, only subunits ATP6V1B1, ATP6V1B2, and ATP6V1E2, previously identified as B1, B2, and E subunits, have been described in the rat epididymis. Here, we report the localization of V-ATPase subunit isoforms ATP6V1A, ATP6V1C1, ATP6V1C2, ATP6V1G1, ATP6V1G3, ATP6V0A1, ATP6V0A2, ATP6V0A4, ATP6V0D1, and ATP6V0D2, previously labeled A, C1, C2, G1, G3, a1, a2, a4, d1, and d2, in epithelial cells of the rat epididymis and vas deferens. Narrow and clear cells showed a strong apical staining for all subunits, except the ATP6V0A2 isoform. Subunits ATP6V0A2 and ATP6V1A were detected in intracellular structures closely associated but not identical to the TGN of principal cells and narrow/clear cells, and subunit ATP6V0D1 was strongly expressed in the apical membrane of principal cells in the apparent absence of other V-ATPase subunits. In conclusion, more than one isoform of subunits ATP6V1C, ATP6V1G, ATP6V0A, and ATP6V0D of the V-ATPase are present in the epididymal and vas deferens epithelium. Our results confirm that narrow and clear cells are well fit for active proton secretion. In addition, the diverse functions of the V-ATPase may be established through the utilization of specific subunit isoforms. In principal cells, the ATP6V0D1 isoform may have a physiological function that is distinct from its role in proton transport via the V-ATPase complex.

Association of mouse sorting nexin 1 with early endosomes.

Sorting nexin 1 (SNX1) is a protein that binds to the cytoplasmic domain of plasma membrane receptors. We found that mouse sorting nexin 1 (SNX1) (521 amino acid residues) could partially rescue a yeast vam3 mutant defective in docking/fusion of vacuolar membranes. In mammalian cells, SNX1 is peripherally associated with membrane structures and localized immunochemically with EEA1, a marker protein of early endosomes. These results suggest that SNX1 regulates endocytic trafficking of plasma membrane proteins in early endosomes. Gel filtration of cell lysates and the purified recombinant protein, together with two-hybrid analysis, indicated that SNX1 self-assembles into a complex of approximately 300 kDa.

Mouse Atp6f, the gene encoding the 23-kDa proteolipid of vacuolar proton translocating ATPase.

The 23-kDa proteolipid subunit of mouse vacuolar-type proton-translocating ATPase (V-ATPase) was predicted to be a hydrophobic polypeptide of 205 amino acid residues with five putative transmembrane segments. It exhibits sequence similarity to Vma16p of Saccharomyces cerevisiae and vha-4 of Caenorhabdittis elegans (83 and 84%, respectively). Southern blot analysis indicated that the proteolipid is encoded by a single gene, Atp6f, in the mouse genome. Atp6f was mapped to approximately 55 cM on chromosome 4, and its genomic organization is similar to that of the human gene: 8 exons separated by 7 introns, with boundaries matching the GT-AG rule. RNA blotting demonstrated that Atp6f is transcribed as 1.0- and 1.8-kb mRNAs in multiple tissues to varying degrees. The major transcription initiation sites are at -13 and -58 bp upstream of the translation initiation codon. The epitope-tagged 23-kDa protoelipid was localized in endomembrane organelles in CHO cells, as expected for a component of a vacuolar-type proton pump.

The murine genome contains one functional gene and two pseudogenes coding for the 16 kDa proteolipid subunit of vacuolar H(+)-ATPase.

We cloned the genomic genes encoding the murine 16 kDa subunit (proteolipid, PL16) of vacuolar H(+)-ATPase (V-ATPase) and determined their nucleotide sequences. At least three independent genes were found in the murine genome. One gene consisted of three exons and was largely identical in sequence to that of PL16 cDNA reported previously (Hanada et al., Biochem. Biophys. Res. Commun. 176 (1991) 1062). In the 5'-flanking region of this gene, several possible transcriptional cis-elements were found. TATA and CAAT sequences were not found, which is characteristic for promoters of house-keeping genes. The other two genes identified did not contain introns. One of these genes had an open reading frame that potentially encoded PL16 but contained six amino acid substitutions and a frame-shift mutation that would result in a truncated protein unable to participate in V-ATPase activity. The other gene had the same sequence in the reading frame as that in the cDNA. However, this gene contained a polyA sequence at the same position where polyA is normally added to mRNA. The gene also had 15 bp repetitive sequences near the transcription initiation site and next to the polyA sequence. These observations suggest that this gene may have been generated by the insertion of reverse-transcribed double-stranded cDNA, as is usually observed for pseudogenes. In conclusion, there is a single functional PL16 gene and two pseudogenes in the murine genome. It is unlikely that PL16 isoforms contribute to variation in V-ATPase function.

a4, a unique kidney-specific isoform of mouse vacuolar H+-ATPase subunit a.

The vacuolar-type H+ -ATPase (V-ATPase) translocates protons across membranes. Here, we have identified a mouse cDNA coding for a fourth isoform (a4) of the membrane sector subunit a of V-ATPase. This isoform was specifically expressed in kidney, but not in the heart, brain, spleen, lung, liver, muscle, or testis. Immunoprecipitation experiments, together with sequence similarities for other isoforms (a1, a2, and a3), indicate that the a4 isoform is a component of V-ATPase. Moreover, histochemical studies show that a4 is localized in the apical and basolateral plasma membranes of cortical alpha- and beta-intercalated cells, respectively. These results suggest that the V-ATPase, with the a4 isoform, is important for renal acid/base homeostasis.

Acidic endomembrane organelles are required for mouse postimplantation development.

Vacuolar-type H(+)-ATPase (V-ATPase) plays a major role in endomembrane and plasma membrane proton transport in eukaryotes. We found that the acidic compartments generated by V-ATPase are present from the one-cell stage of mouse preimplantation embryos. Upon differentiation of trophoblasts and the inner cell mass at the blastocyst stage, these compartments exhibited a polarized perinuclear distribution. PL16(-/-) embryos, lacking the V-ATPase 16-kDa proteolipid (c subunit), developed to the blastocyst stage and were implanted in the uterine epithelium, but died shortly thereafter. This mutant showed severe defects in development of the embryonic and extraembryonic tissues at a stage that coincided with rapid cell proliferation. When cultured in vitro, PL16(-/-) blastocysts could hatch and become attached to the surface of a culture dish, but the inner cell mass grew significantly slower and most cells failed to survive for more than 4 days. PL16(-/-) cells showed impaired endocytosis as well as organellar acidification. The Golgi complex became swollen and vacuolated, possibly due to the absence of the luminal acidic pH. These results clearly indicate that acidic compartments are essential for development after implantation.

Upstream regions directing heart-specific expression of the GATA6 gene during mouse early development.

The expression of murine transcription factor GATA6 is restricted to tissues including the heart and gastrointestinal systems during embryogenesis, and is maintained throughout postnatal life. We have characterized the 5' upstream region (6.4 kb) of the mouse GATA6 gene, and identified two closely spaced transcription initiation sites. The flanking sequence lacks a typical TATA-box, and is rich in guanine and cytosine. The role of the 5' upstream region was examined using the lacZ reporter gene in transgenic mice. A construct containing the 5' flanking sequence (4.9 kb), untranslated exon 1 and 1.3 kb intron 1 could drive the gene expression in the embryonic and adult heart regions. Weak expression was also observed in the stomach, liver, and bronchial arch in addition to the cardiac region. Deletion of the 5' upstream region ( approximately 1.2 kb) or intron 1 abolished all this expression, indicating that at least two cis-acting control elements are necessary for heart-specific expression of GATA6 in vivo.

Luminal acidification of diverse organelles by V-ATPase in animal cells.

Eukaryotic cells contain organelles bounded by a single membrane in the cytoplasm. These organelles have differentiated to carry out various functions in the pathways of endocytosis and exocytosis. Their lumina are acidic, with pH ranging from 4.5 to 6.5. This article describes recent studies on these animal cell organelles focusing on (1) the primary proton pump (vacuolar-type H(+)-ATPase) and (2) the functions of the organelle luminal acidity. We also discuss similarities and differences between vacuolar-type H(+)-ATPase and F-type ATPase. Our own studies and interests are emphasized.

Expression and activity of chimeric molecules between human UDP-galactose transporter and CMP-sialic acid transporter.

Human UDP-galactose transporter (hUGT1) and CMP-sialic acid transporter (hCST) are related Golgi proteins with eight putative transmembrane helices predicted by computer analysis. We constructed chimeric molecules in which segments of various lengths from the C- or N-terminus of hUGT1 were replaced by corresponding portions of hCST. The chimeras were transiently expressed in UGT-deficient mutant Lec8 cells, and their UGT activity was assessed by the binding of GS-II lectin to the transfected cells. The replacement of either the N- or C-terminal cytoplasmic segment by that of hCST did not affect the expression or activity of hUGT1. A chimera in which the eighth helix and the C-terminal tail were replaced also retained the UGT activity, indicating that this helix is not involved in the determination of substrate specificity. In contrast, three types of chimeras, in which the first helix, the first and the second helices, and a segment from the seventh helix to the C-terminus were replaced, respectively, were expressed very infrequently in the transfected cells, and had no UGT activity. They are likely folded incorrectly and degraded by a quality-control system, since the amounts of their mRNAs were normal and the proteins were mainly localized in the ER. The first and the seventh helices are important for the stability of the transporter protein.

Human histamine H2 receptor gene: multiple transcription initiation and tissue-specific expression.

We have characterized the genomic structure of 12.3 kb of the 5'-flanking region of the human histamine H2 receptor gene. The multiple transcription initiation sites of the human histamine H2 receptor gene were mapped utilizing the 5'-end cap structure of mRNA. We found that a 85 bp segment (-610-(-)525 bp) immediately upstream of the initiation site exhibits a strong promoter activity in the gastric adenocarcinoma, MNK45, expressing human histamine H2 receptor. A 4.8 kb transcript of the human histamine H2 receptor gene was found in the placenta, spinal cord, lymph node and bone marrow in addition to the previously reported tissues including the heart, brain and stomach, whereas a 1.8 kb transcript was observed in almost all tissues examined. 3'-rapid amplification of cDNA ends revealed the corresponding length of the 3'-untranslated region. These results suggest that the 3'-untranslated region may be involved in the differential expression.

Nucleotide sugar transporters: elucidation of their molecular identity and its implication for future studies.

Nucleotide sugar transporters are mainly located in the Golgi membranes and carry nucleotide sugars, that are produced outside the Golgi apparatus, into the organelle, where they serve as substrates for the elongation of carbohydrate chains by glycosyltransferases. They are thus indispensable for cellular glycoconjugate synthesis and, moreover, may have regulatory roles in producing the structural variety of cellular glycoconjugates. Their occurrence has long been well recognized, but studies on the molecular bases of their strict substrate specificities and modes of action have been hampered by the lack of information on their precise molecular structures. Complementary DNAs encoding several of these transporters were cloned recently, which represented a substantial step forward as to the above mentioned issues. The products of these cDNAs are mutually related hydrophobic proteins consisting of 320-400 amino acid residues with multiple putative transmembrane helix domains, and are located in the Golgi apparatus. This review briefly summarizes the present status of the field of nucleotide sugar transporter research, and also presents an outlook of the study in this field.

Functional expression of the human UDP-galactose transporters in the yeast Saccharomyces cerevisiae.

We describe the functional expression of the putative human Golgi UDP-galactose transporters (hUGT1 and hUGT2) in the yeast Saccharomyces cerevisiae. Both hUGT1 and hUGT2 were expressed under the control of the yeast constitutive GAPDH promoter. The expression level of hUGT1 seemed to be considerably lower than that of hUGT2, although hUGT1 has an amino acid sequence identical to that of hUGT2 except for 5 amino acid residues at the C-terminus. The hUGT product was expressed in the membranes of Golgi and other organellar compartments. The membrane vesicles prepared from the hUGT1- or the hUGT2-expressing yeast cells exhibited UDP-galactose specific transport activity. The apparent Km values of the yeast-expressed hUGT1 and hUGT2 for UDP-galactose were 1.2 and 2 microM, respectively, which were comparable with the Km obtained with mammalian Golgi vesicles. Transport was dependent on temperature and integrity of vesicles, and was inhibited by UMP, as observed with mammalian Golgi vesicles. Our results demonstrate that the previously described hUGT1 and hUGT2 encode the UDP-galactose transporters, rather than regulatory proteins. The development of a convenient yeast expression system should facilitate analysis of the structure-function relationships of the UDP-galactose transporters.

Functional expression of human golgi CMP-sialic acid transporter in the Golgi complex of a transporter-deficient Chinese hamster ovary cell mutant.

We recently described the cloning of putative human CMP-sialic acid transporter (hCST) cDNA [Ishida, N. et al. (1996) J. Biochem. 120, 1074-1078]. The hCST cDNA coded for a hydrophobic protein with an amino acid sequence showing a high degree of similarity (92% identity) to that of murine CMP-sialic acid transporter. In this report, we demonstrate that hCST corrects the CMP-sialic acid transporter-deficient phenotype of CHO-derived Lec2 cells, as judged from the recovery of WGA-sensitivity by transformants, and the recovery of CMP-sialic acid transporting ability by microsomal vesicles prepared from them. A peptide antibody against the C-terminus of the hCST protein detected the cDNA products expressed in the microsomes of the transformants. The subcellular localization of the hCST protein in the Golgi membrane was demonstrated by immunofluorescence microscopy, using the hCST-specific antibody. These results clearly indicate that hCST cDNA encodes the human CMP-sialic acid transporter protein. Plasma membrane-selective permeabilization combined with immunofluorescence microscopy provided strong evidence that the C-terminus of the human CMP-Sia transporter is exposed to the cytosol on the outer surface of the Golgi membrane.

Expression of the human UDP-galactose transporter in the Golgi membranes of murine Had-1 cells that lack the endogenous transporter.

In our previous study, we demonstrated that UDP-galactose transporter cDNAs (hUGT1 and hUGT2) were able to complement the genetic defect of murine Had-1 cells that were deficient in the UDP-galactose transporter, and that the microsomal vesicles isolated from Had-1-transformants, which were obtained through transfection with these cDNAs, had recovered the ability to uptake UDP-galactose [Ishida, N. et al. (1996) J. Biochem. 120, 1074-1078]. In this report, we describe the preparation of peptide antibodies that recognize the hUGT isozymes, and the detection of hUGT proteins expressed in the transformants. The occurrence of the endogenous hUGT1 protein in HeLa cells was also detected. Using the hUGT1-specific antibodies, the subcellular localization of hUGT1 in the Golgi membrane was demonstrated by immunofluorescence microscopy and subcellular fractionation. These studies led us to develop a simple procedure, based on Percoll density gradient centrifugation, for preparing functional Golgi vesicles from the hUGT1-transformed Had-1 cells, that will facilitate future biochemical analyses of the UDP-galactose transporter for the elucidation of its structure-function relationship.

[A case of advanced gastric cancer with carcinomatosa peritonitis effectively treated by 5-FU and low-dose CDDP therapy].

A 66-year-old woman was admitted to our clinic for appetite loss and abdominal distension in August 1995. Endoscopic study revealed an advanced gastric cancer in the upper body of her stomach. Abdominal CT study revealed massive ascites and para-aortic lymph nodal involvement. Cytological study of the ascites revealed class V. She was diagnosed to be in the terminal stage of gastric cancer with carcinomatosa peritonitis. Combination chemotherapy with 5-fluorouracil (5-FU) and low-dose cisplatin (CDDP) was given by continuous intravenous injection of 5-FU 500 mg/day, and intermittent intravenous injection of CDDP 30 mg/week was performed for reduction of the ascites and her complaint. Endoscopic study 6 weeks after starting chemotherapy could not find crater of the gastric cancer but only a shallow ulcerative lesion. The biopsy specimen of that lesion was group III. No ascites and over 50% reduction of the para-aortic lymph node were found by the abdominal CT study. This state persisted over 4 weeks. No myelo-suppression, renal dysfunction or any severe side effect were observed during chemotherapy. Her performance status improved from 3 to 1.

[A case of invasive thymoma made resectable by preoperative chemotherapy].

A 25-year-old male patient of invasive thymoma was referred to our institute on March'95. He had night coughs and on the annual chest X ray exam a large tumor was found in antero-superior mediastinum. At first his tumor was too large to be resected and left brachiocephalic vein was completely occluded. By needle biopsy it was proved to be thymoma, therefore, preoperative chemotherapy was planned. The tumor responded well to the two courses of ADOC (ADM, CDDP, VCR, CAP) therapy and the tumor seemed to become resectable. In June operation was performed and the tumor was resected completely. It appears that in case of stage Ill invasive thymoma, preoperative chemotherapy is one of the choices although it seems unresectable.

[MRSA enteritis following severe gastroenteritis of salmonellosis].

A 62-year-old female patient was given cancer chemotherapy for lymph nodes metastases in the left breast cancer. She was admitted to the hospital because of severe watery diarrhea, in hypovolemic shock, and was diagnosed as suffering from not-typhoidal Salmonella by stool culture. After systemic administration of antibiotic agents, she became well in a few days, but on the 16th hospital day, she had severe watery diarrhea, hypovolemic shock and then cardiac arrest. She was resuscitated immediately. The stool culture revealed methicillin-resistant Staphylococcus aureus (MRSA), type II coagulase, producing TSST-1 and type BC staphylococcal enterotoxin. It was thought that in this case, MRSA enteritis was caused by damage of the intestinal mucosal barrier of the defense mechanism against infection due to salmonellosis and administration of multiple antibiotic agents.

Computed tomography of parosteal osteosarcoma of the rib.

The computed tomography findings in a case of radically resected parosteal osteosarcoma of the rib are reported. Computed tomography disclosed a huge ossifying mass with lobulated structure, ossifying rim, linear lucent zone between tumor and cortex, destruction of the affected rib cortex, and intramedullary involvement. These findings are specific to advanced parosteal osteosarcoma.